WO2023226921A9 - Récepteur antigénique chimérique bispécifique ciblant bcma-cd19 et son utilisation - Google Patents

Récepteur antigénique chimérique bispécifique ciblant bcma-cd19 et son utilisation Download PDF

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WO2023226921A9
WO2023226921A9 PCT/CN2023/095470 CN2023095470W WO2023226921A9 WO 2023226921 A9 WO2023226921 A9 WO 2023226921A9 CN 2023095470 W CN2023095470 W CN 2023095470W WO 2023226921 A9 WO2023226921 A9 WO 2023226921A9
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sequence
bcma
seq
cells
amino acid
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PCT/CN2023/095470
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WO2023226921A1 (fr
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张超
张其猛
白大勇
吕璐璐
周立
王永增
张云龙
丁伟
路佳兴
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合源康华医药科技(北京)有限公司
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Publication of WO2023226921A1 publication Critical patent/WO2023226921A1/fr
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Definitions

  • This application relates to the field of biomedicine, specifically to a bispecific chimeric antigen receptor targeting BCMA-CD19 and its application.
  • myeloma defined as a malignant proliferation of plasma cells in the bone marrow, is the second most common hematological malignancy, accounting for 1% of all cancers. Studies have shown that multiple myeloma is more common among people over 60 years old and its incidence has increased steadily in recent years. For most patients, multiple myeloma is incurable and eventually develops into relapsed/refractory multiple myeloma. The survival period of patients with relapsed/refractory multiple myeloma who are ineffective against existing multiple myeloma treatments (such as immunomodulators, proteasome inhibitors, and antibody drugs) is only about 13 months.
  • BCMA B Cell Maturation Antigen
  • BCMA is a transmembrane glycoprotein that belongs to the tumor necrosis factor receptor family. BCMA is highly expressed on multiple myeloma cells and not on most other cells. Malignant tumor plasma cells usually express higher levels of BCMA than normal plasma cells. Upregulation of BCMA promotes the growth of multiple myeloma cancer cells, while downregulation of BCMA can inhibit the growth of multiple myeloma cancer cells.
  • multiple myeloma as a B-cell lineage tumor, generally does not express CD19 molecules, so CD19 is usually not used as a target for multiple myeloma treatment.
  • CD19 is usually not used as a target for multiple myeloma treatment.
  • studies in the literature suggesting that some trace amounts of drug-resistant and relapsed multiple myeloma clones also have a CD19 + phenotype.
  • Chimeric Antigen Receptor is the core component of CAR cell therapy, which can include a targeting part (for example, a part that binds to Tumor-Associated Antigen (TAA)), hinge region, trans membrane region and intracellular domain.
  • TAA Tumor-Associated Antigen
  • CAR-T cell immunotherapy is considered to be one of the most promising methods to conquer tumors.
  • CAR-T cells use genetic modification methods to make T cells express CAR proteins. This CAR protein has the ability to recognize intact proteins on the membrane surface without relying on antigen presentation, thereby causing T cell activation and functional effects.
  • This application provides a bispecific chimeric antigen receptor targeting BCMA-CD19 and its application.
  • the inventor constructed multiple bispecific chimeric antigen receptor expression vectors targeting BCMA-CD19 and prepared target Using BCMA-CD19 bispecific CAR-T cells, it was also verified at the cellular level that BCMA-CD19 bispecific CAR-T cells have good tumor suppressive functions.
  • a bispecific chimeric antigen receptor targeting BCMA-CD19 which includes an extracellular antigen recognition domain, a hinge region, a transmembrane region and an intracellular domain; wherein: the extracellular antigen recognition domain includes an anti- BCMA extracellular antigen recognition domain and anti-CD19 extracellular antigen recognition domain;
  • the anti-BCMA extracellular antigen recognition domain includes BCMA VH and BCMA VL, wherein the amino acid sequences of the BCMA VH complementary determining regions CDR1, CDR2, and CDR3 respectively include SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:
  • the amino acid sequence shown in 3 the amino acid sequences of BCMA VL complementarity determining region CDR1, CDR2, and CDR3 include the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 respectively.
  • the anti-CD19 extracellular antigen recognition domain includes CD19 VH and CD19 VL, wherein the amino acid sequences of the CD19 VH complementarity determining regions CDR1, CDR2, and CDR3 respectively include as follows
  • the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, and the amino acid sequences of CD19 VL complementary determining regions CDR1, CDR2, and CDR3 include SEQ ID NO:10 and SEQ ID NO:11 respectively.
  • the BCMA VH sequence includes the amino acid sequence shown in SEQ ID NO: 13
  • the BCMA VL sequence includes the amino acid sequence shown in SEQ ID NO: 14 sequence.
  • the BCMA VH sequence includes the amino acid sequence shown in SEQ ID NO: 15
  • the BCMA VL sequence includes the amino acid sequence shown in SEQ ID NO: 16 sequence.
  • the CD19 VH sequence includes the amino acid sequence shown in SEQ ID NO: 17
  • the CD19 VL sequence includes the amino acid sequence shown in SEQ ID NO: 18 sequence.
  • the extracellular antigen recognition structure of the bispecific chimeric antigen receptor includes any one selected from the following structures: CD19 VL sequence-first linkage Sequence - CD19 VH sequence - 2nd connection sequence - BCMA VL sequence - 3rd connection sequence - BCMA VH sequence, BCMA VL sequence - 4th connection sequence - BCMA VH sequence - 5th connection sequence - CD19 VL sequence - 6th connection sequence - CD19 VH sequence, BCMA VL sequence - 7th junction sequence - CD19 VL sequence - 8th junction sequence - CD19 VH sequence - 9th junction sequence - BCMA VH sequence, and CD19 VL sequence - 10th junction sequence - BCMA VL sequence - The 11th connection sequence - BCMA VH sequence - the 12th connection sequence - CD19 VH sequence.
  • the extracellular antigen recognition domain of the bispecific chimeric antigen receptor includes any one selected from the following structures: BCMA VL sequence-No. 7 Junction Sequence - CD19 VL Sequence - 8th Junction Sequence - CD19 VH Sequence - 9th Junction Sequence - BCMA VH Sequence, and CD19 VL Sequence - 10th Junction Sequence - BCMA VL Sequence - 11th Junction Sequence - BCMA VH Sequence - 12th Linker sequence - CD19 VH sequence.
  • the first linking sequence, the second linking sequence, the third linking sequence, the fourth linking sequence, the fifth linking sequence, the sixth linking sequence, and the seventh linking sequence , the 8th connection sequence, the 9th connection sequence, the 10th connection sequence, the 11th connection sequence, and the 12th connection sequence are independently selected from one or more of the following sequences: SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO:36.
  • the extracellular antigen recognition domain of the bispecific chimeric antigen receptor includes the amino acids shown in SEQ ID NO: 19 or SEQ ID NO: 20 sequence.
  • the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8 ⁇ ; optionally, the hinge region The amino acids of the region are derived from CD8 ⁇ ; further optionally, the amino acid sequence of the hinge region includes the amino acid sequence shown in SEQ ID NO: 21.
  • the transmembrane region is derived from CD3, CD4, CD7, CD8 ⁇ , CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70
  • the amino acid sequence of the transmembrane region is derived from CD8 ⁇ ; further optionally, the amino acid sequence of the transmembrane region includes the amino acid sequence shown in SEQ ID NO: 22.
  • the intracellular domain includes an intracellular signaling region; optionally, it also includes a costimulatory signaling region.
  • the intracellular signaling region is derived from CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, FcR ⁇ , FcR ⁇ , CD66d, DAP10, One or more of DAP12 and Syk; optionally, the intracellular signaling region is derived from CD3 ⁇ ; further optionally, the amino acid sequence of the intracellular signaling region includes as shown in SEQ ID NO: 23 amino acid sequence.
  • the costimulatory signaling region is derived from CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, One, two or more than three of LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, MyD88; optionally, the costimulatory signaling region is derived from CD28 or 4-1BB; Further optionally, the amino acid sequence of the costimulatory signaling region includes the amino acid sequence shown in SEQ ID NO: 24.
  • the above-mentioned dual-specific chimeric antigen receptor also includes a guide peptide located at the N-terminus of the amino acid sequence of the chimeric antigen receptor; optionally, the guide peptide is derived from CD8 ⁇ ; further, it can Optionally, the amino acid sequence of the guide peptide includes the amino acid sequence shown in SEQ ID NO: 25.
  • the dual-specific chimeric antigen receptor includes the amino acid sequence shown in SEQ ID NO: 28 or SEQ ID NO: 29.
  • the present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned dual-specific chimeric antigen receptor.
  • the nucleotide sequence encoding the bispecific chimeric antigen receptor includes:
  • the present application also provides a vector comprising the above-mentioned isolated nucleic acid molecule.
  • the above vector is an expression vector; in some embodiments, the vector is a viral vector; in some embodiments, the vector is a lentiviral vector.
  • the present application also provides an engineered immune effector cell, which contains the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, or the above-mentioned vector.
  • the engineered immune effector cells are selected from T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), multipotent Stem cells, T cells differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, induced pluripotent stem cells (iPSC), T cells differentiated from induced pluripotent stem cells (iPSC-T), Induced pluripotent stem cells differentiate into one or more of NK cells (iPSC-NK) and embryonic stem cells.
  • NK cells natural killer cells
  • PBMC cells peripheral blood mononuclear cells
  • multipotent Stem cells T cells differentiated from pluripotent stem cells
  • NK cells differentiated from pluripotent stem cells induced pluripotent stem cells (iPSC)
  • iPSC-T T cells differentiated from induced pluripotent stem cells
  • iPSC-NK Induced pluripotent stem cells differentiate into one or more of NK cells
  • the engineered immune effector cells are T lymphocytes; optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes. Lymphocytes.
  • This application also provides a pharmaceutical composition, which includes the above-mentioned engineered immune effector cells and pharmaceutically acceptable excipients.
  • pharmaceutically acceptable excipients include protective agents.
  • pharmaceutically acceptable excipients include cell cryopreservation solution.
  • the pharmaceutical composition is an intravenous injection.
  • the present application also provides the use of the above-mentioned chimeric antigen receptor, isolated nucleic acid molecule, vector or engineered immune effector cell in the preparation of medicines for treating diseases or conditions related to the expression of BCMA.
  • the disease or condition associated with the expression of BCMA is cancer; optionally, the cancer is multiple myeloma; further optionally, the cancer is refractory or relapsed Multiple myeloma.
  • the disease or disorder associated with expression of BCMA may be an autoimmune disease.
  • the autoimmune disease may be selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.
  • the present application also provides a method for treating diseases or conditions related to the expression of BCMA, comprising the following steps: applying an effective amount of the above-mentioned engineered immune effector cells or pharmaceutical compositions to patients with a disease or disorder related to the expression of BCMA. Subjects in need of disease or condition.
  • the disease or condition associated with the expression of BCMA is cancer; optionally, the cancer is multiple myeloma; further optionally, the cancer is refractory or relapsed Multiple myeloma.
  • the disease or disorder associated with expression of BCMA may be an autoimmune disease.
  • the autoimmune disease may be selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.
  • the administration is by intravenous injection.
  • the administration method is to administer an effective amount of engineered immune effector cells or a pharmaceutical composition to the subject in a single injection.
  • the effective amount of engineered immune effector cells or pharmaceutical composition is a dose of 1 ⁇ 10 5 to 1 ⁇ 10 7 cells/kg.
  • the present application also provides the above-mentioned engineered immune effector cells or the above-mentioned pharmaceutical composition for treating diseases or conditions related to the expression of BCMA.
  • the disease or condition associated with the expression of BCMA is cancer; optionally, the cancer is multiple myeloma; further optionally, The cancer is refractory or relapsed multiple myeloma.
  • the disease or disorder associated with the expression of BCMA may be an autoimmune disease.
  • the autoimmune disease can be selected from the following: systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, severe disease Myasthenia and autoimmune hemolytic anemia.
  • Figure 1 shows a schematic structural diagram of various BCMA-CD19 bispecific CARs, CD19 CARs, and BCMA CARs in Example 1 of the present application.
  • Figure 2A shows the CAR expression on the surface of CAR-T cells of two complete CARs (i.e., CD19-2A-BCMA and BCMA-2A-CD19) connected to the self-cleaving polypeptide T2A in Example 2 of the present application (6 days after infection) ;
  • the left picture in Figure 2A is the CD19-2A-BCMA group
  • the middle picture in Figure 2A is the BCMA-2A-CD19 group
  • the right picture in Figure 2A is the UTD group (T cells without CAR transduction)
  • Figure 2B to Figure 2E represent The CAR expression on the surface of the four bispecific CAR-T cells (i.e., Tan CD19-BCMA cells, Tan BCMA-CD19 cells, Loop CD19-BCMA cells, and Loop BCMA-CD19 cells) in Example 2 of the present application (after infection 6 days); among them: Figure 2B is the Tan CD19-BCMA group, Figure 2C is the Tan BCMA-CD19 group, Figure 2D is the Loop CD19-BCMA group
  • Figures 3A-3C show the cytokine release of various BCMA-CD19 bispecific CARs, CD19 CARs, and BCMA CARs after being activated by positive target cells in Example 4 of the present application; wherein: Figure 3A is the IL-2 of each group Figure 3B shows the release of IFN- ⁇ in each group, and Figure 3C shows the release of TNF- ⁇ in each group. Among them, Figure 3A, Figure 3B and Figure 3C all contain two dotted boxes.
  • the five columns from left to right represent UTD cells, TanCD19 -Cytokine release from BCMA cells, TanBCMA-CD19 cells, LoopCD19-BCMA cells, LoopBCMA-CD19 cells and BCMA cells after activation by K562-BCMA cells; in the second dotted box in Figure 3A, Figure 3B and Figure 3C , the five columns from left to right represent the cytokine release of UTD cells, TanCD19-BCMA cells, TanBCMA-CD19 cells, LoopCD19-BCMA cells, LoopBCMA-CD19 cells and CD19 cells after activation by K562-CD19 cells.
  • Figure 4A- Figure 4C shows the killing effect of various BCMA-CD19 bispecific CAR, CD19 CAR, BCMA CAR cells on different target cells in Example 5 of the present application; wherein: Figure 4A is the NALM6 group, and NALM6 is CD19+BCMA -Target cells; Figure 4B is the MM.1S group, MM.1S is BCMA+CD19- target cells; Figure 4C is the NALM6-KO CD19 group, NALM6-KO CD19 is a negative target cell control that knocks out CD19 and does not express BCMA. .
  • Figures 5A to 5D show the continued proliferation of CAR-T cells in each group after multiple rounds of antigen stimulation in Example 6 of the present application;
  • Figure 5A is the proliferation of CD3+ cells stimulated with MM.1S cells
  • Figure 5B is The proliferation of CD3+ cells stimulated with NALM6 cells
  • Figure 5C shows the proliferation of CAR+ cells stimulated with MM.1S cells
  • Figure 5D shows the proliferation of CAR+ cells stimulated with NALM6 cells.
  • CAR Chimeric Antigen Receptor
  • extracellular antigen recognition domains for example, binding to tumor-associated antigens (Tumor-Associated Antigen) , part of TAA), hinge region, transmembrane region and intracellular domain.
  • CAR-T (Chimeric Antigen Receptor T) cell immunotherapy is considered to be one of the most promising methods to conquer tumors.
  • CAR-T cells use genetic modification methods to make T cells express CAR proteins. This CAR protein has the ability to recognize intact proteins on the membrane surface without relying on antigen presentation, thereby causing T cell activation and functional effects.
  • extracellular antigen recognition domain refers to the antigen recognition domain (Antigen Recognition Domain, ARD).
  • CAR cell therapy products such as CAR-T cells
  • the antigen recognition domain has been derived from the single antigen of an antibody.
  • Chain variable region Single Chain Variable Fragment, abbreviated as scFv
  • TCR single Chain Variable Fragment
  • VLR variable lymphocyte receptors
  • the scFv includes the antibody heavy chain variable region (VH region) and the light chain variable region (VL region). They are connected by a peptide chain, such as: 18
  • the connecting sequence GSTGSSGKPGSGEGSTKG consists of amino acids.
  • scFv antibodies targeting two or more targets include VH regions and VL regions targeting different targets.
  • the different regions are connected directly or indirectly through connecting sequences, and their arrangement can be in any of the following forms: Target 1 VL - Target 1 VH - Target 2 VL - Target 2 VH, Target 2 VL - Target 2 VH - Target 1 VL - Target 1 VH, Target 1 VL - Target 2 VL - Target 2 VH-Target 1 VH, Target 2 VL-Target 1 VL-Target 1 VH-Target 2 VH, the above "-" represents connection through the connecting sequence.
  • the term "specific recognition and/or binding” refers to the recognition and/or binding between the CAR and the specific target with greater affinity, avidity, easier, and greater affinity than the CAR binding to other targets. and/or bind the target for a greater duration.
  • hinge region refers to the connecting segment between the extracellular antigen recognition domain and the transmembrane domain. This region allows the CAR to recognize the antigen by giving the antigen recognition domain a certain range of activity.
  • the hinge regions currently used are mainly derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8 ⁇ .
  • the typical hinge region also contains some residues that participate in CAR dimerization and help enhance antigen sensitivity.
  • transmembrane region refers to the transmembrane domain that connects the intracellular and extracellular components of the CAR structure. Different transmembrane domains can affect the expression and stability of CAR to a certain extent, but they are not directly involved in signal transmission. They can improve downstream signal transmission through interactions.
  • the transmembrane region may be derived from one or more of CD3, CD4, CD7, CD8 ⁇ , CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70.
  • intracellular domain includes intracellular signaling regions and may also include costimulatory signaling regions.
  • intracellular signaling region refers to the activation of at least one normal effector function of a CAR-expressing immune effector cell.
  • the intracellular signaling region can be derived from one or more of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, FcR ⁇ , FcR ⁇ , CD66d, DAP10, DAP12, and Syk.
  • the term "costimulatory signaling region" exists because in addition to stimulation by antigen-specific signals, many immune effector cells require costimulation to promote cell proliferation, differentiation, and survival, as well as activation of cells. Effector functions.
  • the CAR may also include one or more costimulatory signaling domains, wherein the costimulatory signaling domains may be derived from CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4- One, two or more of 1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3 and MyD88.
  • isolated generally means obtained from the natural state by artificial means. If an "isolated" substance or ingredient occurs in nature, it may be that the natural environment in which it is located has changed, or that the substance has been separated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the high purity of the same polynucleotide or polypeptide isolated from this natural state is called isolation. of.
  • isolated does not exclude substances that have been artificially or synthetically obtained from their natural state by artificial means, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.
  • guide peptide refers to the short peptide before the extracellular antigen recognition domain (such as scFv sequence), which functions to guide the recombinant protein synthesized in the cell to be exported to the outside of the cell.
  • extracellular antigen recognition domain such as scFv sequence
  • Commonly used guide peptides include human CD8 ⁇ signal peptide or human GM-CSF receptor ⁇ signal peptide.
  • BCMA B cell maturation antigen, a member of the tumor necrosis factor receptor superfamily. Human BCMA is expressed almost exclusively on plasma cells and multiple myeloma cells. BCMA may be a suitable tumor antigen target for immunotherapeutics against multiple myeloma. However, due to the heterogeneity of specific antigens on the surface of multiple myeloma cells, the selection of its antigen target is not necessarily a single one. By selecting appropriate targets, the anti-tumor activity of CAR-T cells can be optimized.
  • the "CD19" molecule is currently the main target for the treatment of hematological tumors derived from B lymphocytes, and is also a hot spot in CAR-T cell therapy research. Most malignant tumors derived from B cells express CD19 molecules on their cell surfaces. As a B-cell lineage tumor, multiple myeloma generally does not express CD19 molecules. Therefore, CD19 is usually not used as a target for multiple myeloma treatment. However, there are also studies in the literature suggesting that some trace amounts of drug-resistant and relapsed multiple myeloma clones also have a CD19 + phenotype.
  • dual-target CAR-T products as long as one tumor antigen target is recognized, CAR-T cells can be activated to prevent tumor antigen escape.
  • dual-target CAR immune cells have the following advantages: 1. Fewer immune cells are required, and preparation is convenient and cost-saving; 2. From the perspective of medication In terms of safety, the safety and operability of using one product are much higher than using two products.
  • linking sequence generally refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links together any structure/region of the chimeric antigen receptor of the invention.
  • the linking sequence can be composed of different amino acid residues (such as glycine and serine) so that adjacent protein domains can move freely relative to each other. Longer linker sequences can be used when it is desired to ensure that two adjacent domains do not interfere with each other spatially.
  • isolated nucleic acid molecule generally refers to an isolated form of a nucleotide, deoxyribonucleotide or ribonucleotide of any length, which may be isolated from its natural environment or a synthetic analog .
  • gene transduction/transfection methods mainly include viral and non-viral methods.
  • viral and non-viral methods For example: through gamma retroviral vectors, lentiviral vectors, adenovirus-associated viral vectors, plasmid DNA-dependent vectors, transposon-dependent gene transfer, and mRNA-mediated gene transduction.
  • vector generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed.
  • the vector can transform, transduce or transfect the host cell so that the genetic material elements it carries can be expressed in the host cell.
  • vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); phages such as lambda phage or M13 phage and animal viruses, etc.
  • the types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses.
  • Viruses (such as SV40).
  • a vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication site. Vectors may also contain components that facilitate entry into cells, such as viral particles, liposomes, or protein coats, but they are not the only ones.
  • transposon refers to a discontinuous segment of DNA that has the ability to migrate and carry genetic information between chromosomal sites, such as the Sleeping Beauty SB system and the PB system derived from lepidopteran insects.
  • electroporation can also be used to transduce mRNA into T cells.
  • Immune effector cells generally refers to cells that participate in immune responses, such as promoting immune effector responses.
  • Immune effector cells may be selected from the following groups: T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T lymphocytes differentiated from pluripotent stem cells, pluripotent stem cells differentiated into NK cells, induced pluripotent stem cells (iPSC), induced pluripotent stem cells differentiated into T cells (iPSC-T), induced pluripotent stem cells differentiated into NK cells (iPSC-NK) and One or more types of embryonic stem cells.
  • NK cells natural killer cells
  • PBMC cells peripheral blood mononuclear cells
  • pluripotent stem cells T lymphocytes differentiated from pluripotent stem cells
  • pluripotent stem cells differentiated into NK cells induced pluripotent stem cells (iPSC), induced pluripotent stem cells differentiated into T cells (iPSC-T),
  • the term "pharmaceutical composition” generally refers to a pharmaceutical composition suitable for administration to a patient, which may contain the immune effector cells described in this application, and may also contain one or more pharmaceutically acceptable excipients, such as : One or more of carriers, protective agents, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and preservatives.
  • pharmaceutically acceptable excipients include protective agents, such as cell cryopreservation solutions.
  • the pharmaceutical composition of the present application is a cell suspension or cryopreserved cells thereof.
  • subject generally refers to a human or non-human animal, including but not limited to mouse, rat, cat, dog, rabbit, horse, pig, cow, sheep, or monkey.
  • the term "about” generally refers to a range of fluctuations above or below the specified value that is acceptable to those skilled in the art, such as: within the range of ⁇ 0.5%-10%, such as 0.5 above or below the specified value. %, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, Changes within the range of 9%, 9.5% or 10%.
  • Chimeric antigen receptor nucleic acid, vector, immune effector cell, pharmaceutical composition
  • the present application provides a bispecific chimeric antigen receptor targeting BCMA-CD19, which includes an extracellular antigen recognition domain, a hinge region, a transmembrane region and an intracellular domain; wherein: the extracellular The antigen recognition domain includes an anti-BCMA extracellular antigen recognition domain and an anti-CD19 extracellular antigen recognition domain;
  • the anti-BCMA extracellular antigen recognition domain includes BCMA VH and BCMA VL, wherein the amino acid sequences of the BCMA VH complementary determining regions CDR1, CDR2, and CDR3 respectively include SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:
  • the amino acid sequence shown in 3 the amino acid sequences of BCMA VL complementarity determining region CDR1, CDR2, and CDR3 include the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 respectively.
  • CDR classification rules include Kabat, AbM, Chothia, Contact, and IMGT. These rules are well known to those skilled in the art. When applying the website that implements these rules, just enter the VH and VL sequences and select the corresponding rules. CDR sequences based on different rules can be obtained. In this application, IMGT rules are used to classify CDRs, but those skilled in the art should understand that the protection scope of this application covers combinations of CDR sequences obtained by analyzing using different rules.
  • the anti-CD19 extracellular antigen recognition domain includes CD19 VH and CD19 VL, wherein the amino acid sequences of the CD19 VH complementarity determining regions CDR1, CDR2, and CDR3 respectively include such as SEQ ID NO: 7, SEQ ID NO: 8.
  • the amino acid sequence shown in SEQ ID NO:9, the amino acid sequences of the CD19 VL complementary determining regions CDR1, CDR2, and CDR3 include the amino acids shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12 respectively. sequence.
  • the BCMA VH sequence includes the amino acid sequence set forth in SEQ ID NO: 13
  • the BCMA VL sequence includes the amino acid sequence set forth in SEQ ID NO: 14.
  • the BCMA VH sequence includes the amino acid sequence set forth in SEQ ID NO: 15, and the BCMA VL sequence includes the amino acid sequence set forth in SEQ ID NO: 16.
  • the CD19 VH sequence includes the amino acid sequence set forth in SEQ ID NO:17
  • the CD19 VL sequence includes the amino acid sequence set forth in SEQ ID NO:18.
  • the application also includes the substitution, deletion, addition and/or insertion of one or more amino acids in the amino acid sequence of any of the above-mentioned dual-specific chimeric antigen receptors, and it has an effect equivalent to the above-mentioned Activity of any chimeric antigen receptor.
  • the amino acids in the FR region of the VH and VL sequences can be substituted so that the CDR region of the modified antibody can still retain a suitable antigen-binding site. Therefore, this application naturally Including different amino acid sequences obtained by humanizing the FR regions in the VH and VL sequences based on the above-mentioned CDRs of this application.
  • the extracellular antigen recognition domain of the dual-specific chimeric antigen receptor includes any one selected from the following structures: CD19 VL sequence-1st connection sequence-CD19 VH sequence-2nd connection Sequence - BCMA VL sequence - 3rd connection sequence - BCMA VH sequence, BCMA VL sequence - 4th connection sequence - BCMA VH sequence - 5th connection sequence - CD19 VL sequence - 6th connection sequence - CD19 VH sequence, BCMA VL sequence - 7th linker sequence - CD19 VL sequence - 8th linker sequence - CD19 VH sequence - 9th linker sequence - BCMA VH sequence, and CD19 VL sequence - 10th linker sequence - BCMA VL sequence - 11th linker sequence - BCMA VH sequence - No. 12 connection sequence-CD19 VH sequence.
  • the extracellular antigen recognition domain of the dual-specific chimeric antigen receptor includes any one selected from the following structures: BCMA VL sequence-7th linkage sequence-CD19 VL sequence-8th linkage Sequence - CD19 VH sequence - 9th junction sequence - BCMA VH sequence, and CD19 VL sequence - 10th junction sequence - BCMA VL sequence - 11th junction sequence - BCMA VH sequence - 12th junction sequence - CD19 VH sequence.
  • the 1st connection sequence, the 2nd connection sequence, the 3rd connection sequence, the 4th connection sequence, the 5th connection sequence, the 6th connection sequence, the 7th connection sequence, the 8th connection sequence, the 9th connection sequence , the 10th connection sequence, the 11th connection sequence, and the 12th connection sequence are independently selected from one or more of the following sequences: SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36.
  • the extracellular antigen recognition domain of the dual-specific chimeric antigen receptor includes the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 20.
  • the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8 ⁇ ; optionally, the amino acid sequence of the hinge region is derived from CD8 ⁇ ; further, it can be Optionally, the amino acid sequence of the hinge region includes the amino acid sequence shown in SEQ ID NO: 21.
  • the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8 ⁇ , CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70; optionally, The amino acid sequence of the transmembrane region is derived from CD8 ⁇ ; further optionally, the amino acid sequence of the transmembrane region includes the amino acid sequence shown in SEQ ID NO: 22.
  • the intracellular domain includes an intracellular signaling region; optionally, it also includes a costimulatory signaling region; further optionally, the intracellular signaling region is derived from CD3 ⁇ , CD3 ⁇ , one or more of CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, FcR ⁇ , FcR ⁇ , CD66d, DAP10, DAP12, and Syk; further optionally, the intracellular signaling region is derived from CD3 ⁇ ,
  • the amino acid sequence of the intracellular signaling region includes the amino acid sequence shown in SEQ ID NO: 23.
  • the costimulatory signaling region is derived from CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, One, two or more of NKG2D, DAP10, B7-H3 and MyD88; optionally, the costimulatory signaling region is derived from CD28 or 4-1BB; further optionally, the costimulatory signaling region
  • the amino acid sequence includes the amino acid sequence shown in SEQ ID NO:24.
  • a guide peptide located at the N-terminus of the chimeric antigen receptor amino acid sequence is also included; optionally, wherein the guide peptide is derived from CD8 ⁇ ; further optionally, the amino acid sequence of the guide peptide Contains the amino acid sequence shown in SEQ ID NO:25.
  • the dual-specific chimeric antigen receptor includes the amino acid sequence set forth in SEQ ID NO: 28 or SEQ ID NO: 29.
  • the present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned dual-specific chimeric antigen receptor.
  • the nucleotide sequence encoding the bispecific chimeric antigen receptor comprises:
  • Vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes derived from P1 (PAC); phages such as lambda phage or M13 phage and animal viruses, etc. .
  • the types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses.
  • retroviruses including lentiviruses
  • adenoviruses such as lentiviruses
  • adeno-associated viruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses papillomaviruses
  • papillomaviruses papillomaviruses.
  • Viruses such as SV40
  • the vector is an expression vector; optionally, the vector is a viral vector; further optionally, the vector is a lentiviral vector.
  • the present application also provides an engineered immune effector cell, which contains the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, or the above-mentioned vector.
  • the engineered immune effector cells are selected from the group consisting of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, and T lymphocytes differentiated from pluripotent stem cells.
  • NK cells differentiated from pluripotent stem cells
  • iPSC induced pluripotent stem cells
  • iPSC-T T cells differentiated from induced pluripotent stem cells
  • iPSC-NK differentiated from induced pluripotent stem cells
  • iPSC-NK induced pluripotent stem cells
  • iPSC-T induced pluripotent stem cells
  • iPSC-NK differentiated from induced pluripotent stem cells
  • embryonic stem cells embryonic stem cells.
  • the engineered immune effector cells are T lymphocytes; optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes.
  • the surface of the engineered immune effector cells may express or have chimeric antigen receptors described herein.
  • this application also provides a pharmaceutical composition, which includes the above-mentioned engineered immune effector cells and pharmaceutically acceptable excipients.
  • Pharmaceutically acceptable excipients include: one or more of carriers, protective agents, stabilizers, and diluents.
  • pharmaceutically acceptable excipients include protective agents, such as cell cryopreservation solutions.
  • the pharmaceutical composition is a cell suspension or cryopreserved cells thereof.
  • the pharmaceutical composition is an intravenous injection.
  • the present application also provides a method for preparing engineered immune effector cells, which includes the following steps: transducing the vector described in the application into the immune effector cells.
  • the engineered immune effector cells are selected from the group consisting of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, and T lymphocytes differentiated from pluripotent stem cells.
  • NK cells differentiated from pluripotent stem cells
  • iPSC induced pluripotent stem cells
  • iPSC-T T cells differentiated from induced pluripotent stem cells
  • iPSC-NK differentiated from induced pluripotent stem cells
  • iPSC-NK induced pluripotent stem cells
  • iPSC-T induced pluripotent stem cells
  • iPSC-NK differentiated from induced pluripotent stem cells
  • embryonic stem cells embryonic stem cells.
  • the engineered immune effector cells are T lymphocytes; optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes.
  • the present application also provides the preparation of the bispecific chimeric antigen receptor, the isolated nucleic acid molecule, the vector and/or the engineered immune effector cell described in the present application.
  • the disease or disorder associated with expression of BCMA is cancer; optionally, the cancer is multiple myeloma; further optionally, the cancer is refractory or relapsed multiple myeloma tumor.
  • the disease or disorder associated with expression of BCMA may be an autoimmune disease.
  • the autoimmune disease may be selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.
  • the present application also provides a method for treating a disease or disorder associated with the expression of BCMA, the method comprising the steps of: administering an effective dose to a subject in need of treating a disease or disorder associated with the expression of BCMA.
  • the disease or disorder associated with expression of BCMA is cancer; optionally, the cancer is multiple myeloma; further optionally, the cancer is refractory or relapsed multiple myeloma tumor.
  • the disease or disorder associated with expression of BCMA may be an autoimmune disease.
  • the autoimmune disease may be selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.
  • the administration can be by different means, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the method of administration may be by intravenous injection to the subject.
  • an effective dose of engineered immune effector cells or pharmaceutical compositions can be administered to the subject in a single time, or in divided doses within a certain period, such as once a week, Once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every 3-6 months.
  • the dosage may be different for different indications; the dosage may also be different for patients with different severity of illness.
  • the administered dose may range from 1 ⁇ 10 5 CAR positive T cells/kg to 1 ⁇ 10 7 CAR positive T cells/kg, for example, 1 ⁇ 10 5 CAR positive T cells/kg to 1 ⁇ 10 6 CAR positive T cells/kg, 1 ⁇ 10 6 CAR-positive T cells/kg to 1 ⁇ 10 7 CAR-positive T cells/kg, 0.5 ⁇ 10 6 CAR-positive T cells/kg, 0.6 ⁇ 10 6 CAR-positive T cells /kg, 0.7 ⁇ 10 6 CAR-positive T cells/kg, 0.8 ⁇ 10 6 CAR-positive T cells/kg, 0.9 ⁇ 10 6 CAR-positive T cells/kg, 1.0 ⁇ 10 6 CAR-positive T cells/kg , 1.1 ⁇ 10 6 CAR-positive T cells/kg, 1.2 ⁇ 10 6 CAR-positive T cells/kg, 1.3 ⁇ 10 6 CAR-positive T cells/kg, 1.4 ⁇ 10 6 CAR-positive T cells/kg, 1.5
  • the subjects may include humans and non-human animals.
  • the subject may include, but is not limited to, mouse, rat, cat, dog, horse, pig, cow, sheep, rabbit, or monkey.
  • the present application also provides the chimeric antigen receptor, the isolated nucleic acid molecule, the vector and/or the engineered immune effector cell, which can be used to treat patients with Diseases or conditions associated with expression of BCMA.
  • the disease or disorder associated with expression of BCMA may include a non-solid tumor, optionally, the non-solid tumor is a hematological tumor.
  • the disease or disorder associated with expression of BCMA may include multiple myeloma.
  • the multiple myeloma is relapsed or refractory multiple myeloma.
  • the following examples are only to illustrate the chimeric antigen receptor, engineered immune effector cells, preparation methods and uses of the present application, and are not intended to limit the scope of the invention of the present application.
  • the examples do not include a detailed description of traditional methods, such as those used to construct vectors and plasmids, insert genes encoding proteins into such vectors and plasmids, or introduce plasmids into host cells.
  • Such methods are well known to those of ordinary skill in the art and are described in numerous publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual ,2nd edition,Cold Spring Harbor Laboratory Press.
  • BCMA-specific humanized antibody the amino acid sequence of its VH is shown in SEQ ID NO:13, the nucleotide sequence of its VH is shown in SEQ ID NO:37, and the amino acid sequence of its VL is shown in SEQ ID NO:14 is shown, the nucleotide sequence of its VL is shown as SEQ ID NO:38, and the amino acid sequences of its VH CDR1, CDR2, and CDR3 are shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID respectively.
  • the amino acid sequences of its VL CDR1, CDR2, and CDR3 are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 respectively) and a CD19-specific antibody (its VH
  • the amino acid sequence is shown in SEQ ID NO:17
  • the nucleotide sequence of its VH is shown in SEQ ID NO:39
  • the amino acid sequence of its VL is shown in SEQ ID NO:18
  • the nucleotide sequence of its VL is shown in SEQ ID NO:40 is shown
  • the amino acid sequences of its VH CDR1, CDR2, and CDR3 are shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9 respectively
  • the amino acid sequences of its VL CDR1, CDR2, and CDR3 are respectively As shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12), we hope to obtain a better bispecific CAR structure.
  • BCMA-CD19 bispecific CAR Taking into account changes in the order of the target BCMA and CD19, and the order of VH and VL in the extracellular antigen recognition domain of each target, there are many options for the structural design of the BCMA-CD19 bispecific CAR. We chose to screen candidate BCMA-CD19 bispecific CAR structures on the second-generation CAR structure.
  • CD8 ⁇ guide chain is used as the signal peptide (as shown in SEQ ID NO:25), the hinge region (as shown in SEQ ID NO:21) and the transmembrane region (as shown in SEQ ID NO:22).
  • FIG. 21 adopts the structure of CD8 ⁇ , uses 4-1BB as the intracellular costimulatory signal (as shown in SEQ ID NO:24), and CD3 ⁇ as the T cell activation signal (as shown in SEQ ID NO:23).
  • Loop BCMA-CD19 structure CD19 VL-BCMA VL-BCMA VH-CD19 VH;
  • CD19-2A-BCMA structure CD19 CAR-T2A-BCMA CAR (before and after the self-cleaving polypeptide T2A of this structure, there is a complete CD19 CAR structure and BCMA CAR structure respectively.
  • the complete CAR structure refers to the same structure as other groups. Signal peptide, hinge region, transmembrane region, intracellular costimulatory signal region, T cell activation signal region);
  • BCMA-2A-CD19 structure BCMA CAR-T2A-CD19 CAR (before and after the self-cleaving polypeptide T2A of this structure, there is a complete BCMA CAR structure and CD19 CAR structure respectively.
  • the complete CAR structure refers to the same structure as other groups. Signal peptide, hinge region, transmembrane region, intracellular costimulatory signal region, T cell activation signal region);
  • CD19 CAR structure CD19 VL-CD19 VH;
  • BCMA CAR structure BCMA VL-CD19 VH.
  • the above 6 BCMA-CD19 bispecific CAR structures, 1 CD19 CAR structure, and 1 BCMA CAR structure were constructed into modified empty lentiviral vectors (manufacturer: SBI Company, product number: CD500-CD800, such as WO2021/121227
  • the CAR expression vector was obtained (conventional resistance transformation as described in Example 1), and then the CAR expression vector and three packaging plasmids were transfected into 293T cells. After collection and purification, a functional lentiviral vector was obtained.
  • the three packaging plasmids are pMD2.G (purchased from Biovector Company, product number Biovector012259), pMDLg/pRRE (purchased from Biovector Company, product number Biovector012251), and pRSV-Rev (purchased from Biovector Company, product number Biovector012253).
  • the transduction experiment was performed according to conventional methods known to those skilled in the art.
  • the transduction steps are briefly described as follows:
  • PBMC Peripheral blood mononuclear cells
  • the isolated T cells were cultured with complete lymphocyte culture medium (X-VIVO15 medium + 5% FBS + 300 IU/ml IL-2 or X-VIVO15 medium + 5% FBS + 5ng/ml IL-15 + 10ng/ml IL -7) Resuspend to a final concentration of (1 ⁇ 2) ⁇ 10 6 cells/ml, add 5 ⁇ 10 ⁇ l of CD3/CD28 magnetic beads for stimulation, mix well and place in an incubator for culture.
  • the culture conditions are 37 °C + 5% CO 2 , incubation time is at least 24 hours.
  • Tan CD19-BCMA structure Tan BCMA-CD19 structure, Loop CD19-BCMA structure, Loop BCMA-CD19 structure, CD19-2A-BCMA structure, BCMA-2A-CD19 structure, CD19 CAR, and BCMA CAR respectively.
  • T cells After infecting T cells respectively, the obtained T cells were named Tan CD19-BCMA cells, Tan BCMA-CD19 cells, Loop CD19-BCMA cells, Loop BCMA-CD19 cells, CD19-2A-BCMA cells, and BCMA-2A-CD19 cells. , CD19 cells and BCMA cells.
  • the inventors tried to individually stain the bispecific CAR-T cells obtained in Example 1 after 11 days of infection of the T cells (individual staining refers to bispecific CAR-T cells that are only labeled with FITC fluorescently labeled BCMA antigen or PE fluorescently). CD19 antigen is stained alone) and co-staining (co-staining means that bispecific CAR-T cells are stained by both FITC fluorescently labeled BCMA antigen and PE fluorescently labeled CD19 antigen) to select the staining method.
  • the above observations are not limited by the relative positions of the BCMA extracellular antigen recognition domain and the CD19 extracellular antigen recognition domain in the CAR structure.
  • the BCMA antigen is smaller and is easier to overcome the steric hindrance problem in the bispecific CAR structure and bind to the CAR protein. Therefore, in the subsequent experiments of this application, the BCMA antigen detection data is used to characterize BCMA-CD19
  • the positive proportion of bispecific CAR of course, the positive proportion of BCMA-CD19 bispecific CAR can also be characterized using the data of BCMA antigen detection in the co-staining method, but the separate staining method is simpler.
  • Cytokine release experiment 4 types of bispecific CAR-T cells obtained in Example 1 (Tan CD19-BCMA, Tan BCMA-CD19, Loop CD19-BCMA, Loop BCMA-CD19), BCMA CAR-T cells, CD19 CAR-T cells and UTD cells were co-cultured with target cells in X-VIVO15 medium at an effect-to-target ratio of 1:1 for 24 hours.
  • IL-2, IFN- ⁇ , and TNF- ⁇ in the cell supernatants were detected by ELISA.
  • K562 is a double-negative target cell of BCMA and CD19
  • K562-BCMA is a positive target cell that exogenously expresses BCMA but does not express CD19
  • K562-CD19 is a positive target cell that exogenously expresses CD19 but does not express BCMA. Since K562-BCMA cells do not express CD19 antigen, they were not used to detect CD19 CAR-T cells. For the same reason, K562-CD19 cells did not express BCMA antigen, so they were not used to detect BCMA CAR-T cells (in the figure In 3A, Figure 3B, and Figure 3C, the K562-BCMA group and the K562-CD19 group each lack 1 bar).
  • Example 1 -T cells and UTD cells were co-cultured with target cells in X-VIVO15 medium for 4 hours according to different effector-target ratios (0:1, 1:1, 3:1 or 9:1) of effector cells and target cells. , detect the target cell killing ratio by detecting the luciferase activity stably expressed in the target cells.
  • NALM6 human acute lymphoblastic leukemia cells
  • MM.1S human multiple myeloma cells
  • NALM6 - KO CD19 is a negative target cell control that knocks out CD19 and does not express BCMA.
  • the cell killing results are shown in Figure 4A- Figure 4C: In Figure 4A, Tan CD19-BCMA cells, Tan BCMA-CD19 cells, Loop CD19-BCMA cells, Loop BCMA-CD19 cells, and CD19 CAR-T cells all responded to endogenous NALM6, a positive target cell expressing CD19, has a good killing effect.
  • BCMA CAR-T cells and UTD cells do not have a killing effect on NALM6, a positive target cell expressing CD19 endogenously.
  • Tan CD19-BCMA cells, Tan BCMA -CD19 cells, Loop CD19-BCMA cells, Loop BCMA-CD19 cells, and BCMA CAR-T cells all have good killing effects on MM.1S, a positive target cell that expresses endogenous BCMA.
  • CD19 CAR-T cells and UTD cells The positive target cell MM.1S that endogenously expresses BCMA has no killing effect; in Figure 4C, all cells have no killing effect on the negative target cell control that knocks out CD19 and does not express BCMA.
  • Antigen stimulation can activate CAR-T cells and cause CAR-T cell proliferation. However, continued activation of T cells will lead to cell exhaustion. The proliferation ability and effector function of exhausted T cells will be reduced.
  • BCMA-CD19 bispecific The proliferation of CD3+ cells (i.e., the proliferation of T cells), the proliferation of CAR+ cells, and the proportion of CAR+ cells after multiple rounds of antigen stimulation experiments of CAR-T cells verify the continued proliferation of BCMA-CD19 bispecific CAR-T cells. sex.
  • the four bispecific CAR-T cells (Tan CD19-BCMA, Tan BCMA-CD19, Loop CD19-BCMA, Loop BCMA-CD19), BCMA CAR-T cells, and CD19 CAR obtained in Example 1 were -The CAR-positive proportion of T cells was adjusted by UTD to a level consistent with the group of CAR-T cells with the lowest CAR-positive proportion, including Tan CD19-BCMA cells, Tan BCMA-CD19 cells, Loop CD19-BCMA cells, Loop BCMA- The CAR positive ratio of CD19 cells and BCMA CAR-T cells is based on the BCMA antigen detection data, and the CAR positive ratio of CD19 CAR-T cells is based on the CD19 antigen detection data.
  • each group of CAR-T cells were co-cultured with positive target cells in a 24-well plate at an effective-to-target ratio of 1:2, with 2 ml of X-VIVO15 culture medium per well, and each group of cells was repeated 3 times. hole.
  • the positive target cells were MM.1S and NALM6 respectively, which is equivalent to using BCMA and CD19 as antigens for multiple rounds of stimulation to test the impact of multiple rounds of stimulation with different antigens on the continued proliferation of BCMA-CD19 bispecific CAR-T cells. Since MM.1S cells do not express CD19 antigen, they were not used to detect CD19 CAR-T cells. For the same reason, NALM6 cells did not express BCMA antigen, so they were not used to detect BCMA CAR-T cells.
  • CD3 antibody Manufacturer: BioLegend, Cat. No.: 300312
  • BCMA antigen BCMA antigen
  • CD19 antigen standard for CD3 and CAR staining
  • Tan CD19 -BCMA cells Tan BCMA-CD19 cells, Loop CD19-BCMA cells, Loop BCMA-CD19 cells, BCMA CAR-T cells stained with BCMA antigen, CD19 CAR-T cells stained with CD19 antigen
  • flow cytometry Analysis showing the proportion and number of CAR-positive cells among CD3-positive cells.
  • the number of CAR-positive cells among CD3-positive cells can also be calculated based on the conversion of volume multiples (CD3 is a marker that distinguishes whether they are T cells), and then each calculation result can be used to calculate the number of CAR-positive cells among CD3-positive cells.
  • the group then took out a certain amount of CAR-T cells and added corresponding positive target cells according to the effect-to-target ratio of 1:2 for a new round of stimulation, and repeated 3-4 rounds of stimulation.
  • Tan CD19-BCMA, Tan BCMA-CD19, Loop CD19-BCMA, and Loop BCMA-CD19 structures have better expression on the surface of T cells, cytokine release, and cell killing in vitro.
  • the performance; in terms of the sustained proliferation of CAR-T, the Loop CD19-BCMA and Loop BCMA-CD19 structures are significantly better than the Tan CD19-BCMA and Tan BCMA-CD19 structures and perform better.
  • the Loop CD19-BCMA and Loop BCMA-CD19 structures perform relatively close to BCMA CAR-T and CD19 CAR-T in various experimental tests, and have complete functions for dual targets.
  • SEQ ID NO:1 BCMA VH CDR1;
  • SEQ ID NO:2 BCMA VH CDR2;
  • SEQ ID NO:3 BCMA VH CDR3;
  • SEQ ID NO:4 BCMA VL CDR1;
  • SEQ ID NO:5 BCMA VL CDR2 (ETS, Glu Thr Ser);
  • SEQ ID NO:6 BCMA VL CDR3;
  • SEQ ID NO:9 CD19 VH CDR3;
  • SEQ ID NO:11 CD19 VL CDR2 (SAT, Ser Ala Thr);
  • SEQ ID NO:13 BCMA VH sequence (humanized);
  • SEQ ID NO:14 BCMA VL sequence (humanized);
  • SEQ ID NO:15 BCMA VH sequence (rabbit source);
  • SEQ ID NO:16 BCMA VL sequence (rabbit source);
  • SEQ ID NO:17 CD19 VH sequence
  • SEQ ID NO:18 CD19 VL sequence
  • SEQ ID NO:19 Amino acid sequence of scFv in Loop CD19-BCMA structure
  • SEQ ID NO:20 Amino acid sequence of scFv in Loop BCMA-CD19 structure
  • SEQ ID NO:21 Amino acid sequence of hinge region
  • SEQ ID NO:22 Amino acid sequence of transmembrane region
  • SEQ ID NO:23 Amino acid sequence of intracellular signaling region
  • SEQ ID NO:24 Amino acid sequence of costimulatory signaling region
  • SEQ ID NO:25 Amino acid sequence of guide peptide
  • SEQ ID NO:26 Amino acid sequence of Tan CD19-BCMA structure
  • SEQ ID NO:27 Amino acid sequence of Tan BCMA-CD19 structure
  • SEQ ID NO:28 Amino acid sequence of Loop CD19-BCMA structure
  • SEQ ID NO:29 Amino acid sequence of Loop BCMA-CD19 structure
  • SEQ ID NO:30 Amino acid sequence of CD19-2A-BCMA structure
  • SEQ ID NO:31 Amino acid sequence of BCMA-2A-CD19 structure
  • SEQ ID NO:32 Amino acid sequence of CD19 CAR structure
  • SEQ ID NO:33 Amino acid sequence of BCMA CAR structure
  • SEQ ID NO:34 connection sequence
  • SEQ ID NO:35 connection sequence
  • SEQ ID NO:36 connection sequence
  • SEQ ID NO:37 Nucleotide sequence encoding the BCMA VH amino acid sequence shown in SEQ ID NO:13;
  • SEQ ID NO:38 Nucleotide sequence encoding the BCMA VL amino acid sequence shown in SEQ ID NO:14;
  • SEQ ID NO:39 Nucleotide sequence encoding the CD19 VH amino acid sequence shown in SEQ ID NO:17;
  • SEQ ID NO:40 Nucleotide sequence encoding the CD19 VL amino acid sequence shown in SEQ ID NO:18.

Abstract

La présente invention concerne un récepteur antigénique chimérique bispécifique ciblant BCMA-CD19 et son utilisation. Le récepteur antigénique chimérique bispécifique comprend un domaine de reconnaissance d'antigène extracellulaire ; le domaine de reconnaissance d'antigène extracellulaire comprend un domaine de reconnaissance d'antigène extracellulaire anti-BCMA et un domaine de reconnaissance d'antigène extracellulaire anti-CD19 ; le domaine de reconnaissance d'antigène extracellulaire anti-BCMA comprend BCMA VH et BCMA VL, des séquences d'acides aminés de régions déterminant la complémentarité VH BCMA CDR1, CDR2 et CDR3 comprenant respectivement des séquences d'acides aminés telles que représentées par SEQ ID NO : 1, SEQ ID NO : 2 et SEQ ID NO : 3, et des séquences d'acides aminés de régions déterminant la complémentarité BCMA VL CDR1, CDR2 et CDR3 comprennent des séquences d'acides aminés telles que représentées par SEQ ID NO : 4, SEQ ID NO : 5 et SEQ ID NO : 6.
PCT/CN2023/095470 2022-05-24 2023-05-22 Récepteur antigénique chimérique bispécifique ciblant bcma-cd19 et son utilisation WO2023226921A1 (fr)

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